PROJECT SUMMARY Mammalian genome DNA in the cell nucleus is extensively folded to form a complex three-dimensional (3D) chromatin organization, comprising complex and multivalent interplays of chromatin interactions involving DNA, RNA and protein. The 3D genome is arranged so as to facilitate multiple functional interactions, of which our current understanding is limited, but that ultimately serve to regulate gene expression within a cell. An additional layer of complexity is introduced by the observations that the 3D structural and functional interactions of the chromatin folding are not static, but rather dynamic both over time within a given cell, and between cells of the same type. Understanding these complex functional interactions and their variations will be necessary not only for advancing fundamental biological knowledge, but also for providing novel insights into human disease that could lead to new treatment paradigms. The scientific premise of this project is that at any given time, multiple chromatin interactions are occurring at multiple locations through intricate 3D genome organization and that these interactions are mediated, at least in part, by protein and RNA factors. Unfortunately, the limitations of current 3D genome technologies prevent us from precisely revealing this level of functional complexity at the desirable single-molecule resolution. In this proposal we seek to develop a set of single cell and single molecule techniques for studying multiple, complex chromatin interactions involving protein and RNA regulatory factors within the 3D genome organization. The foundation of our strategy lies in a droplet-based and barcode-linked microfluidics system for single cell and single-molecule detection of complex chromatin interactions. We have developed a prototype for analysis of single-molecule chromatin interactions, called ChIA-Drop (Chromatin Interaction Analysis by Droplet sequencing) that works well for a relatively small Drosophila genome. To develop and refine this approach for mammalian cells, and to begin to uncover the interactions that are critical to chromatin topology and genome functions in health and disease, we propose to achieve the following four aims: Aim 1- Based on proof-of-concept of published results for the Drosophila genome we will use human and murine cell lines to make refinements for the larger mammalian genomes. Aim 2 - we will develop a novel dual-indexing strategy (nucleus-specific and chromatin-specific) for single-cell ChIA- Drop analysis (scChIA-Drop), and will apply it to study multiplex chromatin interactions with single-cell and single-molecule resolution in human and in mouse cells. In Aim 3 - we will extend ChlA-Drop to detect multivalent chromatin interactions mediated by protein and RNA factors. Finally, because datasets produced by ChlA-Drop are new data types, in Aim 4 - we will establish robust computational pipelines and tools for decoding chromatin interactions and make them publicly accessible. The development of these advanced ChIA-Drop technologies and tools will enable unprecedented exploration of chromatin interaction biology and advance our understanding of chromatin topology and genome regulatory functions.